Clostridium difficile enteritis is one of the commonest nosocomial infections and a cause of considerable morbidity and occasional death from perforation of megacolon. C. difficile toxin A is the major determinant of intestinal inflammation and secretion. The overall goal of this proposal is to define the structure and function of the toxin A receptor on rabbit enterocytes. Four specific aims are proposed: 1. Biochemical purification of the enterocyte receptor and amino acid sequencing of receptor peptides. 2. Developmental regulation of the toxin A receptor in neonatal intestine. 3. Association of G proteins with the toxin A receptor. 4. Cloning of the receptor cDNA and determination of the amino acid sequence and carbohydrate composition. The toxin A receptor will be solubilized in detergent from rabbit ileal brush borders and purified to homogenity by affinity chromatography and HPLC. The topographic distribution of the receptor in the rabbit intestine and along the crypt-villus axis will be determined by immunohistochemistry with biotinylated toxin A and with antireceptor antibody. The toxin A receptor is absent in infant rabbits, and immature enterocytes are less responsive to the biological effects of the toxin. Therefore, receptor gene expression will be assessed in developing rabbit ileum using cDNA probes to determine if absence of the receptor in newborns is transcriptionally regulated or is related to post-translational modification. The toxin A receptor is closely associated with membrane G proteins which are likely to be involved in cellular mechanisms of the toxin. The interaction of the receptor with G proteins will be studied in membrane fractions from rabbit intestine and in T84 intestinal cell line. Finally, the isolation and characterization of cDNA clones encoding for the purified toxin A receptor will be carried out. Synthetic oligonucleotide probes corresponding to receptor peptide sequences will be used to screen rabbit cDNA libraries and the cDNA insert of the clone will be characterized and sequenced. Sequence analysis will provide information on the structural characteristics of the receptor and will be compared with sequences of other G protein-associated receptors. Transfection of CHO cells with receptor cDNA will be carried out to determine if biologically active receptor is expressed. These studies will provide information of the cellular basis of toxin A-receptor interaction and on the mechanism of action of this important bacterial enterotoxin on the intestine.